Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz-dependent polymerization activity

Nath, Pratiti; Morona, Renato

doi:10.1099/mic.0.000042

Cited by 18 publications

(16 citation statements)

References 35 publications

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“…These membrane-anchored oligosaccharides are then flipped across the IM to the periplasmic face by the Wzx flippase, where they are polymerized by Wzy into polysaccharides with various numbers of RUs, often referred to as O units in the case of O antigens. A Wzz chain length regulator normally interacts with Wzy in a yet-to-be-understood manner to confer a distinctive modal chain length distribution (9)(10)(11)(12)(13). The resultant polysaccharides are then removed from the Und-P moiety, and in the case of O antigens, are attached to lipid A core by the ligase WaaL to give mature LPS.…”

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confidence: 99%

Progress in Our Understanding of Wzx Flippase for Translocation of Bacterial Membrane Lipid-Linked Oligosaccharide

2018

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Translocation of lipid-linked oligosaccharides is a common theme across prokaryotes and eukaryotes. For bacteria, such activity is used in cell wall construction, polysaccharide synthesis, and the relatively recently discovered protein glycosylation. To the best of our knowledge, the Gram-negative inner membrane flippase Wzx was the first protein identified as being involved in oligosaccharide translocation, and yet we still have only a limited understanding of this protein after 3 decades of research. At present, Wzx is known to be a multitransmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varied degrees of preference. In this review, we provide an overview of the major findings for this protein, with a particular focus on substrate preference.

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confidence: 99%

Progress in Our Understanding of Wzx Flippase for Translocation of Bacterial Membrane Lipid-Linked Oligosaccharide

2018

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“…Therefore, PL3 possesses essential positively-charged amino acids throughout which includes a conserved Arg residue that was identified in a large sequence alignment of heterologous Wzy-like proteins. With the observation of essential positive charge in PL3 of Wzy β , it lends support to the role of a proximal periplasmic loop being utilized for substrate recruitment (Islam et al, 2011 , 2013a ; Nath and Morona, 2015 ).…”

Section: Discussionmentioning

confidence: 71%

“…Upon investigation of the net-charges across both Wzy β PL regions, no particular amino acid stretch with notable positive charge was found within PL3 of Wzy β , rather, a net-negative charge was seen. This does not discredit the catch-and-release mechanism as both the Shigella flexerni and Francisella tularensis Wzy proteins have a negatively-charged PL3 but in the case of S. flexneri the Arg residue in the noted RX 15 G motif was found to be essential for function (Kim et al, 2010 ; Nath and Morona, 2015 ). Previously, our lab used a position-based algorithm, Jackhmmer (Finn et al, 2011 ) and discovered distant homologs of Wzy α.…”

Section: Discussionmentioning

confidence: 99%

A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα

et al. 2016

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Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3′ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272, and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyβ utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.

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“…The growing O-antigen is initially made by transfer of an oligosaccharide entity from OS-PP-Und onto another OS-PP-Und acting as the acceptor ( Fig. 4) Moreover, as shown for the S. flexneri Wzy the guanidinium group of arginine residues, studied by mutation to lysine or glutamic acid, of the large periplasmic loops is important for the polymerizing activity but also for the association with Wzz during the polymerization process (vide infra) (97).…”

Section: The Wzx/wzy Pathwaymentioning

confidence: 99%

Lipopolysaccharides of Gram-Negative Bacteria: Biosynthesis and Structural Aspects

Ståhle

Widmalm

2019

TIGG

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Gram-negative bacteria are to a large extent covered by lipopolysaccharide (LPS) anchored in the outer leaflet of their outer membrane. There are presently four described pathways for the O-antigen assembly of LPS, viz., synthase-, Wzk-, ABC-transporterand Wzx/Wzy-dependent pathways, where the latter two are used in Escherichia coli, subject to the O-antigen polysaccharide to be made. NDP-sugar monosaccharides are used by glycosyltransferases in the process of linking sugar residues together in the cytoplasm and depending on the biosynthetic pathway polymerization of the O-antigen takes place either in the cytoplasm (ABCtransporter pathway) or in the periplasm, where an oligosaccharide anchored in the inner membrane is flipped to the periplasmic side to this end (Wzx/Wzy-dependent pathway). Additions of sugars to form side-chains on the O-antigen may also occur in the periplasmic space. The degree of polymerization of the O-antigen is regulated to give a modal distribution, i.e., a narrow distribution around the most probable chain-length. The O-antigen is subsequently conjugated to the lipid A-core to form the LPS, which is transported across the periplasmic region by an ATP-driven mechanism as part of an LPS transport (Lpt) system. By using predictions of NDPmonosaccharide pathways and glycosyltransferase function it is shown how O-antigen structure can be elucidated rapidly by the computer program CASPER using bioinformatics data in conjunction with unassigned NMR data of the polysaccharide.For the bacterium the LPS acts as an effective permeability barrier to cationic antimicrobial peptides, enhances intracellular survival and contributes to the evasion of the immune defenses by mimicry of host molecules (16).An LPS molecule is a lipoglycan and contains three structural GLYCO REVIEW

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Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz-dependent polymerization activity

Cited by 18 publications

References 35 publications

Progress in Our Understanding of Wzx Flippase for Translocation of Bacterial Membrane Lipid-Linked Oligosaccharide

Progress in Our Understanding of Wzx Flippase for Translocation of Bacterial Membrane Lipid-Linked Oligosaccharide

A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα

Lipopolysaccharides of Gram-Negative Bacteria: Biosynthesis and Structural Aspects

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